Plasmacytoid Dendritic cells (pDCs) are the most potent producers of interferons, which are critical antiviral cytokines. pDC development is, however, compromised following a viral infection, and this phenomenon, as well as its relationship to conventional (c)DC development is still incompletely understood. By using lymphocytic choriomeningitis virus (LCMV) infection in mice as a model system, we observed that DC progenitors skewed away from pDC and toward cDC development during in vivo viral infection. Subsequent characterization of the transcriptional and epigenetic landscape of fms-like tyrosine kinase 3 + (Flt3 + ) DC progenitors and follow-up studies revealed increased apoptosis and reduced proliferation in different individual DC-progenitors as well as a profound type I interferon (IFN-I)-dependent ablation of pre-pDCs, but not pre-DC precursors, after both acute and chronic LCMV infections. In addition, integrated genomic analysis identified altered activity of 34 transcription factors in Flt3 + DC progenitors from infected mice, including two regulators of Glucocorticoid (GC) responses. Subsequent studies demonstrated that addition of GCs to DC progenitors led to downregulated pDC-primed-genes while upregulating cDC-primed-genes, and that endogenous GCs selectively decreased pDC, but not cDC, numbers upon in vivo LCMV infection. These findings demonstrate a significant ablation of pre-pDCs in infected mice and identify GCs as suppressors of pDC generation from early progenitors. This provides a potential explanation for the impaired pDC development following viral infection and links pDC numbers to the hypothalamic–pituitary–adrenal axis.

Type I Interferons (IFN-I) are central to host protection against viral infections, with plasmacytoid dendritic cells (pDC) being the most significant source, yet pDCs lose their IFN-I production capacity following an initial burst of IFN-I, resulting in susceptibility to secondary infections. The underlying mechanisms of these dynamics are not well understood. Here we find that viral infection reduces the capacity of pDCs to engage both oxidative and glycolytic metabolism. Mechanistically, we identify lactate dehydrogenase B (LDHB) as a positive regulator of pDC IFN-I production in mice and humans; meanwhile, LDHB deficiency is associated with suppressed IFN-I production, pDC metabolic capacity, and viral control following infection. In addition, preservation of LDHB expression is sufficient to partially retain the function of otherwise exhausted pDCs, both in vitro and in vivo. Furthermore, restoring LDHB in vivo in pDCs from infected mice increases IFNAR-dependent, infection-associated pathology. Our work thus identifies a mechanism for balancing immunity and pathology during viral infections, while also providing insight into the highly preserved infection-driven pDC inhibition.

Type I Interferons (IFN-I) are central to host protection against viral infections 1 . While any cell can produce IFN-I, Plasmacytoid Dendritic Cells (pDCs) make greater quantities and more varieties of these cytokines than any other cell type 2 . However, following an initial burst of IFN- I, pDCs lose their exceptional IFN-I production capacity and become "exhausted", a phenotype that associates with enhanced susceptibility to secondary infections 3-5 . Despite this apparent cost for the host, pDC exhaustion is conserved across multiple species and viral infections, but the underlying mechanisms and the potential evolutionary advantages are not well understood. Here we characterize pDC exhaustion and demonstrate that it is associated with a reduced capacity of pDCs to engage both oxidative and glycolytic metabolism. Mechanistically, we identify lactate dehydrogenase B (LDHB) as a novel positive regulator of pDC IFN-I production in mice and humans, show that LDHB deficiency is associated with suppressed IFN-I production, pDC metabolic capacity, and viral control following a viral infection, and demonstrate that preservation of LDHB expression is sufficient to partially restore exhausted pDC function in vitro and in vivo . Furthermore, restoring LDHB in vivo in exhausted pDCs increased IFNAR dependent infection- associated pathology. Therefore, our work identifies a novel and conserved mechanism for balancing immunity and pathology during viral infections, while also providing insight into the highly preserved but previously unexplained phenomenon of pDC exhaustion.

During a microbial infection, responding CD8+ T cells give rise to effector cells that provide acute host defense and memory cells that provide sustained protection. An alternative outcome is exhaustion, a state of T cell dysfunction that occurs in the context of chronic infections and cancer. Although it is evident that exhausted CD8+ T (TEX) cells are phenotypically and molecularly distinct from effector and memory CD8+ T cells, the factors regulating the earliest events in the differentiation process of TEX cells remain incompletely understood. Here, we performed single-cell RNA-sequencing and single-cell ATAC-sequencing of CD8+ T cells responding to LCMV-Armstrong (LCMV-Arm) or LCMV-Clone 13 (LCMV-Cl13), which result in acute or chronic infections, respectively. Compared to CD8+ T cells that had undergone their first division in response to LCMV-Arm (Div1ARM) cells, CD8+ T cells that had undergone their first division in response to LCMV-Cl13 (Div1CL13) expressed higher levels of genes encoding transcription factors previously associated with exhaustion, along with higher levels of Ezh2, the catalytic component of the Polycomb Repressive Complex 2 (PRC2) complex, which mediates epigenetic silencing. Modulation of Ezh2 resulted in altered expression of exhaustion-associated molecules by CD8+ T cells responding to LCMV-Cl13, though the specific cellular and infectious contexts, rather than simply the level of Ezh2 expression, likely determine the eventual outcome. Taken together, these findings suggest that the differentiation paths of CD8+ T cells responding to acute versus chronic infections may diverge earlier than previously appreciated.

Mitotically stable random monoallelic gene expression (RME) is documented for a small percentage of autosomal genes. We developed an in vivo genetic model to study the role of enhancers in RME using high-resolution single-cell analysis of natural killer (NK) cell receptor gene expression and enhancer deletions in the mouse germline. Enhancers of the RME NK receptor genes were accessible and enriched in H3K27ac on silent and active alleles alike in cells sorted according to allelic expression status, suggesting enhancer activation and gene expression status can be decoupled. In genes with multiple enhancers, enhancer deletion reduced gene expression frequency, in one instance converting the universally expressed gene encoding NKG2D into an RME gene, recapitulating all aspects of natural RME including mitotic stability of both the active and silent states. The results support the binary model of enhancer action, and suggest that RME is a consequence of general properties of gene regulation by enhancers rather than an RME-specific epigenetic program. Therefore, many and perhaps all genes may be subject to some degree of RME. Surprisingly, this was borne out by analysis of several genes that define different major hematopoietic lineages, that were previously thought to be universally expressed within those lineages: the genes encoding NKG2D, CD45, CD8α, and Thy-1. We propose that intrinsically probabilistic gene allele regulation is a general property of enhancer-controlled gene expression, with previously documented RME representing an extreme on a broad continuum.

Natural killer (NK) cells are a major subset of innate immune cells that are essential for host defense against pathogens and cancer. Two main classes of inhibitory NK receptors (NKR), KIR and CD94/NKG2A, play a key role in suppressing NK activity upon engagement with tumor cells or virus-infected cells, limiting their antitumor and antiviral activities. Here, we find that single-chain NKR antagonists linked to a VHH that binds the cell surface phosphatase CD45 potentiate NK and T activities to a greater extent than NKR blocking antibodies alone in vitro. We also uncovered crosstalk between NKG2A and Ly49 that collectively inhibit NK cell activation, such that CD45-NKG2A and CD45-Ly49 bispecific molecules show synergistic effects in their ability to enhance NK cell activation. The basis of the activity enhancement by CD45 ligation may reflect greater antagonism of inhibitory signaling from engagement of MHC I on target cells, combined with other mechanisms, including avidity effects, tonic signaling, antagonism of weak inhibition from engagement of MHC I on non-target cells, and possible CD45 segregation within the NK cell-target cell synapse. These results uncover a strategy for enhancing the activity of NK and T cells that may improve cancer immunotherapies.

Viruses that cause chronic infections, including Human Immunodeficiency Virus (HIV), Hepatitis B virus (HBV), and Hepatitis C virus (HCV), infect more than 500 million people worldwide and represent a major health burden. Chronic viral infections suppress immune responses to promote their own persistence. Plasmacytoid Dendritic Cells (pDCs) are an innate cell type which are specialized to produce exceptionally high levels of type I interferon (IFN-I) in response to viral infection, however in the context of chronic viral infection they lose the capacity to produce IFN-I. The metabolic program of pDCs has previously been implicated to be crucial for their function, but it is unclear how chronic infection modifies pDC metabolism. We demonstrate that chronic infection stimulates metabolic reprogramming in pDCs. Specifically, chronic infection is associated with sustained suppression of respiratory metabolism in pDCs. Furthermore, we demonstrate that respiratory metabolism in pDCs is highly correlated to their ability to produce cytokine, and that byproducts of respiratory metabolism (mitochondrial derived reactive oxygen species) can promote cytokine production in pDCs. These data suggest that chronic infection drives changes in pDC metabolism that could be associated with infection driven pDC exhaustion.

Dendritic cells (DC) play a central role in immune responses and can broadly subdivided into conventional (c) and plasmacytoid (p) DCs. We and others have described several adaptations of DCs and their progenitors during acute and chronic infections, including impaired DC development, maturation and altered cytokine production. To understand the underlying mechanisms we determined the transcriptional and chromatin landscapes of bone marrow (BM) DC progenitors from lymphocytic choriomeningitis virus (LCMV) infected mice, via RNA-Seq and ATAC-Seq, respectively. Initial analysis indicated that infection induced multiple alterations in gene signatures, including type-I-interferon signaling and metabolic pathways, as well as changes in chromatin accessibility, which were more enriched within intergenic regions. We next used the Taiji algorithm, which integrates chromatin accessibility and gene expression to assess the global importance of transcription factors (TFs) at the systems level. This analysis identified 11 known DC regulators and 24 TFs with no previous connection to DC biology. Knock-down experiments revealed that Glucocorticoid Modulatory Element Binding Protein 1 (Gmeb1), which was predicted to exhibit increased activity in progenitors from LCMV-infected mice, suppressed DC development and maturation. On the other hand, Zinc Finger Protein 524 (Zfp524), whose activity was predicted to be reduced in progenitors from LCMV-infected mice, promoted pDC cytokine production while inhibiting the same function in cDCs. These results highlight two novel TFs that regulate DC development and/or function, deepening our understanding of DC biology and providing potential new targets for DC-based immunotherapies.

ABSTRACT Chronic viral infections represent a major challenge to the host immune response, and a unique network of immunological elements, including cytokines, are required for their containment. By using a model persistent infection with the natural murine pathogen lymphocytic choriomeningitis virus clone 13 (LCMV Cl13) we investigated the role of one such cytokine, interleukin-27 (IL-27), in the control of chronic infection. We found that IL-27 receptor (IL-27R) signaling promoted control of LCMV Cl13 as early as days 1 and 5 after infection and that il27p28 transcripts were rapidly elevated in multiple subsets of dendritic cells (DCs) and myeloid cells. In particular, plasmacytoid DCs (pDCs), the most potent type 1 interferon (IFN-I)-producing cells, significantly increased il27p28 in a Toll-like receptor 7 (TLR7)-dependent fashion. Notably, mice deficient in an IL-27-specific receptor, WSX-1, exhibited a pleiotropy of innate and adaptive immune alterations after chronic lymphocytic choriomeningitis virus (LCMV) infection, including compromised NK cell cytotoxicity and antibody responses. While, the majority of these immune alterations appeared to be cell extrinsic, cell-intrinsic IL-27R was necessary to maintain early pDC numbers, which, alongside lower IFN-I transcription in CD11b + DCs and myeloid cells, may explain the compromised IFN-I elevation that we observed early after LCMV Cl13 infection in IL-27R-deficient mice. Together, these data highlight the critical role of IL-27 in enabling optimal antiviral immunity early and late after infection with a systemic persistent virus and suggest that a previously unrecognized positive-feedback loop mediated by IL-27 in pDCs might be involved in this process. IMPORTANCE Persistently replicating pathogens, such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, represent major health problems worldwide. These infections impose a long-term challenge on the host immune system, which must be heavily and continuously regulated to keep pathogen replication in check without causing fatal immunopathology. Using a persistently replicating rodent pathogen, LCMV, in its natural host, we identified the cellular sources and effects of one important regulatory pathway, interleukin-27 receptor WSX-1 signaling, that is required for both very early and late restriction of chronic (but not acute) infection. We found that WSX-1 was necessary to promote innate immunity and the development of aberrant adaptive immune responses. This not only highlights the role of IL-27 receptor signaling in regulating distinct host responses that are known to be necessary to control chronic infections, but also positions IL-27 as a potential therapeutic target for their modulation.

Although characterization of T cell exhaustion has unlocked powerful immunotherapies, the mechanisms sustaining adaptations of short-lived innate cells to chronic inflammatory settings remain unknown. During murine chronic viral infection, we found that concerted events in bone marrow and spleen mediated by type I interferon (IFN-I) and Toll-like receptor 7 (TLR7) maintained a pool of functionally exhausted plasmacytoid dendritic cells (pDCs). In the bone marrow, IFN-I compromised the number and the developmental capacity of pDC progenitors, which generated dysfunctional pDCs. Concurrently, exhausted pDCs in the periphery were maintained by self-renewal via IFN-I-and TLR7-induced proliferation of CD4(-) subsets. On the other hand, pDC functional loss was mediated by TLR7, leading to compromised IFN-I production and resistance to secondary infection. These findings unveil the mechanisms sustaining a self-perpetuating pool of functionally exhausted pDCs and provide a framework for deciphering long-term exhaustion of other short-lived innate cells during chronic inflammation.

During chronic infections, sustained cell adaptation via functional exhaustion has been mostly studied in the adaptive immune compartment but much less is known on how innate immune cells adjust to a persistently infectious milieu. Thus, to understand how innate cells adapt to persistent infections, we studied plasmacytoid dendritic cells (pDCs), which specialize in Type I Interferon (IFN-I) production and often become functionally exhausted in chronic settings. Using a murine chronic viral infection model we demonstrated that pDC IFN-I exhaustion is caused by concerted events at central (bone marrow) and local (spleen) compartments. On one hand, undifferentiated bone marrow pDC progenitors exhibited quantitative and qualitative defects and failed to generate functional pDCs ex vivo. Analysis of transcription and chromatin landscapes via RNA-seq and ATAC-seq revealed that IFN-I signaling pathway was enhanced in bone marrow pDC progenitors. Consistently, IFN-I receptor blockade restored the quantitative (albeit not the qualitative) defects of bone marrow pDC progenitors, which was accompanied by restoration of E2-2, a transcription factor critical for pDC development. On the other hand, splenic pDCs received sustained TLR7 signaling, which promoted their maturation but also rendered them unable to produce IFN-I upon ex vivo TLR re-stimulation or in vivo secondary infection. RNA-seq and ATAC-seq analysis in splenic pDCs revealed significant alterations in multiple pathways and transcriptional regulators that are currently being studied. Our work provides an anatomical, cellular and molecular framework to understand how innate immune exhaustion can be triggered and sustained during a chronic viral infection.

The ascarosides, small-molecule signals derived from combinatorial assembly of primary metabolism-derived building blocks, play a central role in Caenorhabditis elegans biology and regulate many aspects of development and behavior in this model organism as well as in other nematodes. Using HPLC-MS/MS-based targeted metabolomics, we identified novel ascarosides incorporating a side chain derived from succinylation of the neurotransmitter octopamine. These compounds, named osas#2, osas#9, and osas#10, are produced predominantly by L1 larvae, where they serve as part of a dispersal signal, whereas these ascarosides are largely absent from the metabolomes of other life stages. Investigating the biogenesis of these octopamine-derived ascarosides, we found that succinylation represents a previously unrecognized pathway of biogenic amine metabolism. At physiological concentrations, the neurotransmitters serotonin, dopamine, and octopamine are converted to a large extent into the corresponding succinates, in addition to the previously described acetates. Chemically, bimodal deactivation of biogenic amines via acetylation and succinylation parallels posttranslational modification of proteins via acetylation and succinylation of L-lysine. Our results reveal a small-molecule connection between neurotransmitter signaling and interorganismal regulation of behavior and suggest that ascaroside biosynthesis is based in part on co-option of degradative biochemical pathways.

The ascarosides, small-molecule signals derived from combinatorial assembly of primary metabolism-derived building blocks, play a central role in C. elegans biology and regulate many aspects of development and behavior in this model organism as well as in other nematodes. Using HPLC-MS/MS-based targeted metabolomics, we identified novel ascarosides incorporating a side-chain derived from succinylation of the neurotransmitter octopamine. These compounds, named osas#2, osas#9, and osas#10, are produced predominantly by L1 larvae, where they serve as part of a dispersal signal, whereas these ascarosides are largely absent from the metabolomes of other life stages. Investigating the biogenesis of these octopamine-derived ascarosides, we found that succinylation represents a previously unrecognized pathway of biogenic amine metabolism. At physiological concentrations, the neurotransmitters serotonin, dopamine, and octopamine are converted to a large extent into the corresponding succinates, in addition to the previously described acetates. Chemically, bimodal deactivation of biogenic amines via acetylation and succinylation parallels posttranslational modification of proteins via acetylation and succinylation of L-lysine. Our results reveal a small-molecule connection between neurotransmitter signaling and interorganismal regulation of behavior and suggest that ascaroside biosynthesis is based in part on co-option of degradative biochemical pathways.

In the model organism Caenorhabditis elegans, a class of small molecule signals called ascarosides regulate development, mating, and social behaviors. Ascaroside production has been studied in the predominant sex, the hermaphrodite, but not in males, which account for less than 1% of wild-type worms grown under typical laboratory conditions. Using HPLC-MS-based targeted metabolomics, we show that males also produce ascarosides and that their ascaroside profile differs markedly from that of hermaphrodites. Whereas hermaphrodite ascaroside profiles are dominated by ascr#3, containing an α,β-unsaturated fatty acid, males predominantly produce the corresponding dihydro-derivative ascr#10. This small structural modification profoundly affects signaling properties: hermaphrodites are retained by attomole-amounts of male-produced ascr#10, whereas hermaphrodite-produced ascr#3 repels hermaphrodites and attracts males. Male production of ascr#10 is population density-dependent, indicating sensory regulation of ascaroside biosynthesis. Analysis of gene expression data supports a model in which sex-specific regulation of peroxisomal β-oxidation produces functionally different ascaroside profiles.